Project Summary/Abstract Dopaminergic (DA) neurons degenerate in Parkinson?s disease (PD) and upon abuse of the addictive stimulant, methamphetamine (METH) and related amphetamine-type stimulants (ATS). The molecular mechanisms involved in DA neuron degeneration are not yet clear, either in PD or ATS neurotoxicity. To develop novel treatments that prevent DA neural degeneration, underlying mechanisms need to be better understood. To study these mechanisms, several in vitro experimental models have been developed. However, none of these models features DA neurons with axon terminals that innervate their proper target, striatal neurons. The presence of striatal neurons is essential because these neurons produce chemo-attractive cues that not only direct the growth of DA axons and but also promote dopamine transporter (DAT) expression in the DA axon terminals. If striatal cues are not available, DA neurons appear to express DAT improperly, in the somatic plasma membrane. Notably, the somatic DAT behaves abnormally ? it is internalized upon an exposure to METH. Such DAT internalization is not observed in vivo in the DA axon terminals innervating striatal neurons. The goal of this project is to develop an improved in vitro cultured DA cell model in which striatal neurons will be innervated by DAT-expressing axon terminals. In the proposed model, primary cultures of ventral mesencephalon (VM) and striatal neurons will be plated nearby on the same substrate, such that the dopaminergic neurons present in the VM cultures can innervate the striatal neurons. The VM and striatal neurons will be obtained from HA-DAT mice. These mice have a hemagglutinin (HA) tag inserted into the DAT. The HA tag does not affect DAT trafficking and activity but allows to fluorescently visualize DAT in live cells. It is expected that during culturing, the cues from the striatal neurons will promote DAT expression and activity in the VM neurites innervating the striatal neurons. The project has two aims. Aim 1: Develop the co-cultures. Aim 2: Characterize DAT expression and activity in the co-cultures. Once developed and validated, the proposed model will be used to study the mechanisms underlying DAT involvement in METH-induced DA neurotoxicity and the mechanisms underlying DA cell degeneration in PD.